FR2950358A1 - SIMPLIFIED NUCLEIC ACID AMPLIFICATION DEVICE AND ITS IMPLEMENTATION METHOD - Google Patents

Abstract

The present invention relates to a disposable device (100) for amplifying at least one target nucleic acid, present in a liquid and biological sample of interest, which consists of a solid body (2), at least one fluidic channel (3) connecting an inlet (4), by which all or part of the sample of interest can be sucked and / or discharged, and an outlet (5), which is itself connected to a means of suction-discharge of said sample of interest, the fluidic channel (3) further comprising the inlet (4) to the outlet (5): • a first compartment (8) containing all or part of the thermostable constituents, • means for mixing (15) constituents with the sample of interest, • a second compartment (9) containing all or part of the non-thermostable constituents, and • additionally at least one zone for heating said sample of interest (6) mixed with said amplification constituents, in order to allow amplification of the target nucleic acid. The invention also proposes an amplification method using such a device. The present invention finds a preferential application in the field of medical diagnosis.

Description

DESCRIPTION

The present invention relates to a disposable device for amplifying at least one target nucleic acid. The present invention also relates to a method of amplifying such a target nucleic acid, which uses a previously cited device. The device, like the method, can be used with any type of amplification technique, such as PCR or a post-transcriptional amplification technique, such as TMA or NASBA.

The state of the art is constituted by the document WOA-99/33559 which relates to an integrated cartridge allowing the manipulation of fluids, but also of the same applicant, the document WO-A-2006/132886, which deals with a method and an apparatus for storing and using beads reagents. This system consists of a compact instrument with four identical and removable modules and a multi-well cartridge that incorporates a multi-port valve. A piston moves liquids from one well to another of the cartridge to mix them. Among the different zones of the cartridge, each of them may either permit: • filtration on a silica matrix to capture, purify and concentrate the sample studied, • lysis which, via glass beads and under the action of ultrasound generate by an instrument, can lyse the cell membranes of the cells present in the sample, • the amplification is carried out by transfer of all or part of the eluate in the zone amplification and detection (PCR) of the cartridge. The cartridge can integrate solid or liquid reagents or both simultaneously.

This system is fully integrated and automated, with a result of short enough time (of the order of one hour). However, because of the chosen concept, the cartridge is relatively complex and expensive. It is therefore not very suitable for a high speed system. In addition, it does not make it possible to perform several tests per sample other than by multiplexing the amplification primers and / or detection probes in the same volume. This makes the development of biological assays much more complex and time consuming with inevitable compromises in detection performance.

Our invention is much better suited to high speeds because the card we protect can be easily manipulated by a PLC in the manner of a simple pipetting cone. In addition its lateral size is reduced which allows to consider a high speed architecture using a fluorescence reading carousel of acceptable diameter for a system designed for high speed. Finally, the elongated design, with a flat surface, makes it easy to move this card within a reading carousel requiring: • to ensure a good thermal contact for the thermal cycles (PCR type) or a constant and homogeneous temperature ( NASBA type, TMA) while allowing said card to be moved by sliding on the incubation blocks, and to perform a fluorescence readout, the card being able to be easily moved in front of the different read heads (different wavelengths) . High speed means a rate of over 300 tests per day, with a low cost per test and a small footprint.

Compared with this system, the present invention makes it possible to have costs per test compatible with routine viral or microbiological diagnosis, at a high rate, but also for tests that are deported to patients, otherwise known as POCT (Point Of Care Testing). ), thanks in particular to the simplicity of the developed consumable, its integrated depipetage function and the reduced volume of amplification reaction, making it possible to reduce the cost per test (the enzymes in molecular biology constitute the major part of this cost per test for the part "reagents" and a decrease in the reaction volume makes it possible to have a reduction proportional to the reduction in volume) (5 pL instead of 50 to 80 pL in these applications).

The state of the art also includes document WO-A-2004/004904 which relates to an apparatus designed to carry out rapid tests in an autonomous and mobile manner, in the context, in particular, of bioterrorism, bacteriological warfare, and POCTs. , device that requires a minimum of manual operations. This system uses a cartridge with input ports connected to a flexible pouch with one or more compartments in which the amplification reagents (PCR) are lyophilized. The fluorescence is read directly through the deformable flexible film.

The pouches are kept under vacuum which allows, after the introduction of the nucleic acid samples via the inlet of the consumable port, to carry out an automatic and calibrated filling and to dissolve the lyophilized reagents prior to the amplification by PCR.

This device is not suitable for performing routine high speed tests. This system of distribution and / or fluidic division by vacuum is effective for single-stage fluidic (filling) protocols that can be used in the case of PCR amplification. But it is poorly suited to nucleic acid amplification process, which require successively resuspend several reagents, as is the case for NASBA or TMA amplifications. This therefore requires adding a valve and maintaining a partial vacuum between the two chambers containing the amplification reagents, resulting in a complexification of the consumable and the associated instrument. In addition the consumable does not allow to aspirate directly, without manual intervention, the sample containing nucleic acids (pipetting) there is no pipetting function. In addition, this device, which combines a rigid part and a flexible pouch part, is not easily manageable in an architecture in which the user can put a sample at any time even during the operation of the instrument, architecture called " Random Access. Our invention, on the other hand, proposes a card which can easily be manipulated by a robot and be used as a cone for pipetting solutions, mixing them, taking up the eluate and sucking it in to effect reactions such as reactions. amplification. The card, according to the present invention, can therefore be used as a pipette cone for making additions, aspirate liquid reagents in tubes, and thus perform the steps prior to the amplification / detection step. With appropriate automation, said card can also take and remove several cones at the same time. For this purpose embodiments, comprising more than one fluidic path, are presented later in this document.

The present invention of the Applicant, by its original design, allows to adapt to both PCR amplification protocol (or RT-PCR) requiring only a reagent (and therefore achievable in a single chamber) or protocol two-step amplification process requiring the separation, in two separate chambers, amplification reagents, as is the case with post-transcriptional amplifications. The invention also solves the problem of architecture of the instrument, by proposing a consumable (or card) that can be used either in a POCT system (a few tests per day) or in a high-speed routine diagnostic system (greater than 300 tests per day) by the integration of the pipetting function, which uses conventional cones (reported or integrated). The state of the art is also constituted for the document WO-A-2007/100500. It is a very POCT-oriented system or the low flow in molecular biology. This easy-to-use system makes it possible to work directly from a drop of blood (some ten pL). The associated instrument is also compact thanks to a combination of actuators making it possible to isolate the compartments of the tubular shaped consumable and thus to allow the transfer and mixing of the liquids in the order defined during the manufacture by the prior filling. consumable. A major disadvantage of this system is its lack of flexibility on the biological protocol to achieve. On the one hand, the volumes of samples and buffers (magnetic nucleic acid capture particles, washing and elution buffers) are fixed by the volume of each compartment of the consumable, as defined in the manufacture of the consumable. . It is known that it is often necessary to resort to the modification of the biological protocol in order to obtain better capture, amplification and nucleic acid detection performance as a function of various parameters, for example the nature of the biological sample treated, the presence or absence of inhibitors (sputum, BAL, plasma, urine, whole blood, etc.). With this state-of-the-art system, the modification of the protocols necessitates the creation each time of a different consumable with different volumes for each compartment and thus of modifying the sealing zones, with a very strong constraint related to the location. zones of implantation of the mobile actuators in the associated instrument (valves and pistons for ur-pressing rupture of said sealing zones). On the other hand, the sample volume remains very small and therefore does not cover all the needs of users, especially those related to tests starting from several milliliters of sample. On the other hand, the flexible object is difficult to manage by a PLC except to have a significant additional cost. It has no pipetting function or flexibility in the choice of elution and wash volumes, and therefore less flexibility in sample preparation.

The applicant has also filed a certain number of documents which may constitute the state of the art, in particular the patent application EP-B-1,187,678, which concerns a device for implementing a card. analysis in which reaction stages and fluid transfers are performed under the effect of internal control means to the card. A problem of this type of device is that it is obliged to operate using valves which consist of deformable elements under the action of an actuator which causes the direct or indirect closure of the channels associated with the valves. The essential problem with this device is the complexity. Thus the presence of valves considerably complicates the production of the analysis card thus formed and strikes its production price, and secondly, there is a need for many external elements (actuators) to enable the actuation of the assembly. said valves.

The present invention proposes to solve the problems highlighted by all the documents of the state of the art mentioned above. To do this, it offers a device that is disposable and that meets a number of technical characteristics. In a particularly advantageous embodiment of the invention, the device is a consumable which is considered as a pipette, carrying the dried or lyophilized reagents necessary for amplification of RNA or DNA targets from a reduced volume of nucleic acids. (5 to 10 μL), which reduces reagent costs and incorporates a valve to eliminate any risk of contamination. It is a slide valve, normally open and closed after location of the reaction volume in the reading zone, when there are no more steps to achieve. This type of device has many unknown qualities of the state of the art: • Ability to realize, from this concept, PLCs in a POCT version (batch processing of 8-24 samples per batch) and in one version of high speed automaton using the same consumable. • Automatic removal, by displacement of the attached tip or an integral part of the device according to the invention, purified nucleic acid volume to reduce the number of manual steps, which therefore simplifies the automation of an automated device using such consumables. • Ability to integrate a PCR or NASBA amplification for the POCT into the same associated instrument. • Simplification of the instrumentation by integrating into the device embedded reagents and lyophilized or dried, which can be dissolved and mixed successively by simple movement within said device whose configuration of the fluidic circuit facilitates this dissolution and mixing. • Ability to perform mono-tests (one set of amplification primers per fluid channel within the device), multiplex tests (with at least two sets of primers per channel) or panel (at least two sets of separate primers in at least two channels) from a common instrumental architecture. • Total automation of the amplification protocol with a low-cost device and associated instrumentation that is compact. • Reduction of the total amplification time (in particular in NASBA) by reduction of the denaturation time compared to a "conventional" amplification (1 minute versus 5 to 10 minutes) due to a heating through the thin film of sealing of the reaction channel, in place of a thicker plastic tube, whose thermal inertia is unfavorable. • No life time constraint of the enzyme compared to conventional automation, the reagents, embedded in said device, remaining dry until their recovery by the liquid sample to be amplified.

The present invention relates to a disposable device for the amplification of at least one target nucleic acid, present in a liquid and biological sample of interest, which consists of a solid body, at least one fluid channel connecting an inlet , by which all or part of the sample of interest can be sucked and / or discharged, and an outlet, which is itself connected to a suction-discharge means of said sample of interest, the fluidic channel comprising in addition to the input to the output: • a first compartment containing all or part of the thermostable constituents necessary for carrying out the amplification, • a means for mixing the constituents with the sample of interest, • a second compartment containing all or part of the non-thermostable constituents necessary for carrying out the amplification, and in addition at least one zone intended for heating said sample of interest mel to said amplification components, to allow amplification of the target nucleic acid. According to one embodiment, the device for the detection of amplicons is characterized in that it furthermore comprises, at the level of the second compartment, all or part of the detection constituents necessary for the detection of amplicons. According to another embodiment, the device, allowing the detection of amplicons, is characterized by the fact that it further comprises at the level of the fluidic channel a third compartment containing all or part of the constituents necessary for the detection of amplicons. Still according to another embodiment, the device further comprises at the level of the fluidic channel a third compartment containing nothing but for the subsequent detection of amplicons in a clean environment. According to an alternative embodiment, the device, described in the previous paragraph, the third compartment is located between the second compartment and the output of the device. Regardless of the embodiment or embodiment, the inlet of the device receives a pipette cone or the pipette tip has a pipette cone configuration. Whatever the embodiment or the preceding embodiment, the suction-discharge device is of piston type such as for example a pipette. Whatever the mode or the preceding embodiment, the section of the channel is constant and the compartments are of a larger section. According to a multi-channel embodiment, the input is in relation with at least two fluidic channels. Still according to a multi-channel embodiment, the output consists of at least two fluidic channels. Whatever the embodiment or the preceding embodiment, the constituents are formed of freeze-dried or dried biological compounds, soluble in the sample of interest. Whatever the mode or the embodiment which precedes, the suction-discharge means is an integral part of the disposable device. According to this latter embodiment, the suction-discharge means consists of a cylinder connected to the fluidic channel and a piston movable within the cylinder 20 manually or via an actuator. Whatever the mode or embodiment of the preceding embodiment, the mixing means is constituted by the fluid channel whose path comprises at least one baffle.

The present invention also provides a method of amplifying at least one target nucleic acid, present in a liquid and biological sample of interest, made within a previously described device, which comprises: (a) aspirating by entering all or part of the sample of interest into the device, (b) moving said sample to dissolve the thermostable amplification constituents, (c) mixing the sample and thermostable constituents, (d) applying a first gradient temperature to denature the nucleic acid of interest, (e) moving the mixture to dissolve non-thermostable amplification constituents, (f) mixing mixture and non-thermostable constituents, and (g) applying at least one second temperature gradient to amplify the denatured nucleic acid. According to one embodiment, the thermostable amplification components of step (b) also contain restriction enzymes, which are not necessarily thermostable, but which allow cleavage at predetermined positions of the nucleic acids of interest, which are deoxyribonucleic acids, prior to the application of the first temperature gradient of step (d). According to another embodiment, which can be used in addition to the previously mentioned mode, the detection of the amplicons consists, after step (g) in: (h) moving the new mixture to dissolve the detection constituents therein. (i) mixing mixture and detection components, and (j) detecting the presence of amplicons. According to another embodiment, which can be used in addition to at least one of the previously mentioned modes, the amplification is a PCR amplification, for which the first temperature gradient is between 90 and 100 ° C., and the second temperature gradients is an alternation of the temperature in three different stages: between 90 and 100 ° C for the first denaturation temperature, preferably around 94 ° C, between 50 and 60 ° C for the second temperature; hybridization, preferably at about 55 ° C, between 70 and 75 ° C for the third polymerization temperature, preferably about 72 ° C. According to another embodiment, which may be used in addition to at least one of the aforementioned modes, the amplification is a post-transcriptional amplification (NASBA or TMA), for which the first temperature gradient is between 60 and 70 ° C, preferably about 65 ° C, and the second temperature gradient is between 40 and 50 ° C for the second polymerization temperature gradient. According to another embodiment, which can be used in addition to at least one of the aforementioned modes, the first temperature gradient is applied at the level of the first compartment and / or the mixing means, and the second gradient or gradients temperature is or are applied at the level of the mixing means and / or the second compartment and / or the third compartment. Still according to another embodiment, which can be used in addition to at least one of the previously mentioned modes, the first temperature gradient is applied for 5 to 20 minutes, preferably 15 minutes, and the second temperature gradient or gradients is or are applied: in the case of a PCR amplification: for the denaturation, for less than one minute, preferably from 2 to 20 seconds, preferably 5 seconds, for the hybridization, for less than one minute, preferably for 2 minutes; at 20 seconds, preferably 5 seconds, and for the polymerization, for less than two minutes, preferably from 5 to 80 seconds, preferentially 10 seconds, in the case of a post-transcriptional amplification, for less than two hours, preferably from 5 to 80 seconds. minutes, and even more preferably about 60 minutes in the case of RNA target nucleic acids, or about 90 minutes in the case of DNA target nucleic acids.

The following terms may be used interchangeably in the singular or the plural. The term "component" means both "reagent", "amplification reagent", "extraction reagent" or "purification reagent" or "raw material" and refers to reagents, such as reaction buffers, the enzymes, the mono-, bi- or triphosphate nucleosides, but also the solvents, the salts necessary for carrying out a reaction for the extraction, purification or enzymatic amplification of a nucleic acid. By "container" or "plastic container" within the meaning of the present invention is meant any container such as tubes, cones or pipette tips, whether plastic (eg Eppendorf type) or glass or "any other material". For the purposes of the present invention, the term "nucleic acid" means a sequence of at least two nucleotides, preferably at least ten nucleotides chosen from the four types of nucleotides of the genetic code, namely if the nucleic acid is a DNA: dAMP (deoxyadenosine 5'-monophosphate), dGMP (deoxyguanosine 5'-monophosphate), dTMP (deoxythymidine 5'-monophosphate), and dCMP (deoxycytidine) '-monophosphate), if the nucleic acid is an RNA: • AMP (adenosine 5'-monophosphate), • GMP (guanosine 5'-monophosphate), • UMP (uridine 5'-monophosphate), and • CMP (cytidine 5'-monophosphate).

The nucleic acid may also optionally comprise at least one inosine and / or at least one modified nucleotide. The term "modified nucleotide" means in the present invention, a nucleotide, for example at least one nucleotide comprising a modified nucleotide base, deoxyuridine, diamino-2,6-purine, bromo-5-deoxyuridine or any other modified base, preferentially with the exception of 5-methyl-cytosine. The nucleic acid can also be modified at the level of the internucleotide linkage, for example phosphorothioates, H-phosphonates or alkylphosphonates, at the level of the backbone such as, for example, the alpha-oligonucleotides (FR-A-2,607,507). or the polyamide nucleic acids (PMA) (Egholm M. et al., J. Am Chem Soc., 1992; 114; 1895-97) or the 2'-O-alkyl-ribonucleotides and / or a 2 'nucleotide O-fluoro and / or a 2'-amine nucleotide and / or an arabinose nucleotide, and LNAs (Sun BW et al., Biochemistry, 2004, Apr. 13, 43 (14): 4160-69). Of the 2'-O-alkyl-ribonucleotides, 2'-O-methyl-ribonucleotides are preferred, but 5-Propinyl Pyrimidine Oligonucleotides (Seitz O., Angewandte Chemie International Edition 1999; 38 (23)) can also be used. Dec .: 3466-69). The term "nucleotide" defines either a ribonucleotide or a deoxyribonucleotide. For the purposes of the present invention, the term "biological sample" or "liquid biological sample", any sample capable of containing nucleic acids. These can be extracted from tissue, blood, serum, saliva, circulating cells of a patient or from a food product, food or environmental origin. The extraction is carried out by any protocol known to those skilled in the art, for example by the isolation method described in patent EPB-0.369.063. For the purposes of the present invention, the term "contaminant" or "contaminating acid" or "contaminating nucleic acid" or "contaminating element" is intended to mean any nucleic acid whose amplification is not desired and which is capable of generating a result. false positive during detection. By "amplification" or "amplification reaction" is meant any nucleic acid amplification technique well known to those skilled in the art, such as PCR (Polymerase Chain Reaction), described in US-A-4,683,195 , US-A-4,683,202 and US-A-4,800,159, and its RT-PCR derivative (Reverse Transcription PCR), in particular in a one-step format, as described in EP-B-0,569,272. Preferably, the PCR is carried out on a single strand with a single pair of primer. LCR (Ligase Chain Reaction), for example disclosed in patent application EP-B-0.201.184, - RCR (Repair Chain Reaction), described in patent application WO-A-90/01069, La 3SR ( Self Sustained Sequence Replication) with patent application WO-A-90/06995, NASBA (Nucleic Acid Sequence-Based Amplification) with patent application WO-A-91/02818, TMA (Transcription Mediated Amplification) with U.S. Patent No. 5,399,491; and RCA (Rolling Circle Amplification) disclosed in US Patent No. 6,576,448. Within the meaning of the present invention, the term "target" or "target nucleic acid" or "target nucleic" or "target of interest" or "nucleic acid of interest", a nucleic acid (an oligonucleotide, a polynucleotide , a nucleic acid fragment, a ribosomal RNA, a messenger RNA, a transfer RNA) to be amplified and / or detected. The target can be extracted from a cell or synthesized chemically. The target may be free in solution or bound to a solid support. The term "liquid and biological sample of interest" means a homogeneous or heterogeneous aqueous solution. By "solid support" is meant particles which may be latex, glass (GPC), silica, polystyrene, agarose, sepharose, nylon, etc. These materials may possibly allow the confinement of magnetic material. It can also be a filter, a film, a membrane or a strip. These materials are well known to those skilled in the art. The target may be a viral, bacterial, fungal or yeast nucleic acid, present in a mixture, in the form of a single or double strand of DNA and / or RNA. . In general, the target is between 50 and 10,000 nucleotides long, but most often it is between 100 and 1000 nucleotides. By "marker" is meant a molecule carried by a nucleotide. The link between the marker and the nucleotide can be done in different ways known to those skilled in the art. Manual coupling is by the use of markers bearing an activated group, typically a carboxyl or a thiol, which are coupled to a modified internal nucleotide carrying the corresponding reactive group (amine or thiol, for example), or on one end of the nucleotide strand modified with these same reactive groups. The automatic coupling is done by the use of phosphoramidites carrying the marker, and then the coupling is done during the automated synthesis of the nucleotide strand, either on one end of the strand or on an internal position, depending on the type of phosphoramidite used. The marker may be a fluorophore or a fluorescent quencher. By "fluorophore" is meant a molecule that emits a fluorescence signal when it is excited by light at a suitable wavelength. The fluorophore may be in particular a rhodamine or a derivative such as Texas Red, a fluorescein or a derivative (for example FAM), a fluorophore of the Alexa family such as Alexa532 and Alexa647, Alexa 405, Alexa 700, Alexa 680, Cy5 or any other fluorophore that is suitable for the measuring device used. The fluorophores available for detection probes are very varied and known to those skilled in the art. For the purposes of the present invention, the term "fluorescein" means an aromatic chemical molecule that emits a fluorescence signal with a maximum emission around 530 nm, when it is excited by light at a wavelength in the vicinity. from 490 to 500 nm, preferably 495 nm. By "fluorescent extinguisher" or "quencher" is meant a molecule that interferes with the fluorescence emitted by a fluorophore. This extinguisher can be chosen from non-fluorescent aromatic molecules, to avoid parasitic emissions. Preferably, said extinguisher is a Dabsyl or a Dabcyl or a "black hole quencherTM" (BHQ) which are non-fluorescent aromatic molecules that prevent the emission of fluorescence when they are physically close to a fluorophore. The fluorescence resonance energy transfer technique (FRET) can also be used as described, for example, in Fluorescent Energy Transfer Nucleic Acid Probes, p. 4, Ed V. V.V. Didenko, Humana Press 2006, ISSN 1064-3745. The quencher may also be selected from fluorescent molecules, such as, for example, TAMRA (carboxyetetramethylrhodamine).

The "three-base detection probe" or "three-base probe", referred to as S3B, is a probe as defined above and which, in addition to the preceding characteristics, consists of a nucleotide sequence of three different types of bases chosen from the group adenine, thymine, guanine, cytosine. It is well understood by those skilled in the art that according to the shape of the probes (Molecular Beacon, see EP95 / 904 104.7, EP96 / 303 544.9 and EP97 / 923 412.7, or 0-probe, see PCT / FR2009 / 051315, etc. ), the probe will consist of a part of a sequence whose nucleotide sequence will be composed of nucleotides of four different types of bases and of a sequence whose nucleotide sequence will be composed of nucleotides of three different types of bases ( sequence for hybridization and detection of amplicons).

In some cases, to improve the hybridization with the amplicons and therefore their detection, the probes according to the invention may occasionally contain uracil instead of thymine. In this case, the probes according to the invention will consist of a nucleotide sequence of four different types of bases (uracil, guanine, adenine, thymine). By "hybridization" is meant the process in which, under appropriate conditions, two single-stranded nucleotide fragments, having all or part of complementary sequences, are capable of forming a double-strand or "duplex" stabilized by hydrogen bonds between the nucleic bases. The hybridization conditions are determined by the stringency, that is to say the rigor and low salinity of the operating conditions. Hybridization is all the more specific as it is carried out with higher stringency. The stringency is defined in particular according to the base composition of a probe / target duplex, as well as by the degree of mismatch between two nucleic acids. The stringency may also be a function of the parameters of the reaction, such as the concentration and type of ionic species present in the hybridization solution, the nature and the concentration of denaturing agents and / or the hybridization temperature. The stringency of the conditions under which a hybridization reaction must be performed will depend primarily on the hybridization probes used. All these data are well known and the appropriate conditions can be determined by those skilled in the art.

The examples and accompanying figures show particular embodiments and can not be considered as limiting the scope of the present invention. FIG. 1 represents a first embodiment of the disposable device according to the invention, which contains a single and only fluidic channel. In this particular configuration, the device is ready to aspirate a biological sample to be treated in which at least one target nucleic acid is likely to be present. FIG. 2 again represents this first embodiment, but in another particular configuration, in which the device is sucking up the biological sample to be treated (not shown in this figure). FIG. 3 represents a second embodiment of the disposable device according to the invention, which contains two fluidic channels in parallel. In this particular configuration, the device is ready to aspirate said biological sample containing at least one target nucleic acid, which may be present.

FIG. 4 also represents this second embodiment, but in another particular configuration, in which the device is sucking up the biological sample to be treated (not shown in this figure).

FIG. 5 represents a third embodiment of the disposable device according to the invention, which contains eight fluidic channels in parallel. In this particular configuration, the device is ready to aspirate said biological sample containing at least one target nucleic acid, which may be present. FIG. 6 represents a section along A-A of FIG. 5 making it possible to better visualize the manner in which the suction and delivery actions are performed within said device. Finally, Figures 7 to 10 show the device according to a variant of the second embodiment, but in use: • Figure 7: The disposable device does not contain the liquid sample of interest. Its tip is just in contact with it which is present in a container. With the tip being in contact with the sample, the pistons are moved to allow aspiration of the sample of interest into the disposable device. In this case the sample is found at the level of the first compartment and the thermostable constituents that it contains. Before the pistons have completed their aspiration at this phase, the disposable device is mounted and / or the container is lowered, so that the device and sample of said container are no longer in contact and that the single channel is filled more than of air, as is the case in this figure 8. The two downstream channels, each containing a liquid column of an aliquot of the sample. Preferably the suction by the pistons is stopped when the device is removed from the remaining sample at the container. FIG. 9: Aspiration of the sample of interest by said continuous pistons and said sample is moved towards the mixing means, where back-and-forth movements allow the mixing of the sample and thermostable constituents to be well mixed. . The sample assembly associated with thermostable constituents is thereafter always called "sample" for reasons of ease of reading and comprehension. • Figure 10: The pistons are moved to allow aspiration of the sample of interest within the disposable device. In this case the sample is found in the second compartment and non-thermostable constituents it contains. • Figure 11: There is no more aspiration, but a discharge of the sample of interest by said pistons and said sample is moved to the mixing means, where back and forth movements allow the good mixing of the sample and non-thermostable constituents. Again, the sample assembly associated with the thermostable and non-thermostable constituents is thereafter always called "sample" for reasons of ease of reading and comprehension. • Figure 12 The pistons are moved to allow the aspiration of the sample of interest within the disposable device. In this case, the sample is found at the level of the third compartment and the constituents necessary for the detection it contains. Figure 13 shows NASBA amplification curves for the state of the art (EasyQ) according to increasing values of the target quantity (see scale from 0 to 10000 in the high position - see reference B). The curves corresponding to the amplification of the HIV target sequence are plotted in the lower position (see reference to the arrow A), the curves corresponding to the calibrator are in the upper position.

FIG. 14 represents NASBA amplification curves for the invention under the same conditions as those applied in FIG. 13 (likewise for the use of the arrows A and B). Note that to improve the readability in this figure, the curves corresponding to the HIV target were expanded by a factor of 5. Thus with the device according to the present invention, the curves corresponding to the target (HIV) vary from 8 to 40 rfu (Relative Fluorescence Unit) approx. The curves of the calibrator ranging from 50 to 250, the graph is not readable if we put the two curves with the same scale. Thus, the HIV curves vary between 40 and 200 and are therefore more readable. Finally, they are relative values that do not prejudge the efficiency of the amplification, it is actually the inflection points of each curve which, they have a value of interpretation. FIG. 15 proposes the variable quantization calculated by the HIV v.2.0 algorithm for the EasyQ instruments (on the left) and on the invention (on the right). Figure 16 relates to the quantization result as a function of the target amount. Finally, FIG. 17 represents a longitudinal section along B-B of FIG. 2 of the device according to the invention.

The present invention is well represented throughout FIGS. 1 to 17. Three embodiments are more particularly shown.

A first embodiment shown in FIGS. 1 and 2 represents a very simple embodiment of the present invention. It consists of a disposable device which is formed of a solid body 2 in which is etched on the surface a circuit or fluidic channel 3. Of course this fluid circuit 3 is delimited by a film of BOPP type (Bi-Oriented PolyPropylen ), not referenced in these two figures, but present with the reference 14 in Figure 17, which prevents the liquid sample from leaving said circuit 3. The fluidic channel 3 comprises on the one hand a through hole 4, called input, located in bottom of each figure and another opening hole, said outlet, located at the top of each figure, which allows the channel 3 to have two openings on the outside. In fact the inlet 4 is formed by a pipette tip, referenced 16, that is to say molded in one piece with the entire body 2. In Figure 1 in pipette tip reported 27 is shown , showing a particular embodiment using a conventional tip. On the other side, the outlet 5 is present within a cylinder 17, which constitutes one of the parts of the discharge suction means 7. The other part of this discharge suction means 7 is constituted by a piston 18 which can slide, within the cylinder 17, moved by an actuator, not represented here, according to the arrow F1, on the one hand, which is a movement of the piston 18 in the cylinder 19 in which the volume of fluid within the circuit fluid 3 decreases, or according to the arrow F2, which is a movement of the piston 18 in the cylinder 19 which is totally different from the previous one, that is to say that it increases the volume of fluid within the fluidic circuit 3. The actuator can act on the piston via a connecting means 21. This system is particularly relevant in the case where it is desired to automate the entire system. In this embodiment of Figures 1 and 2, it is noted that the fluidic circuit is relatively simple since it comprises along the channel 3 of substantially constant section, a first compartment 8, a second compartment 9 and, between the two 8 and 9, a mixing means 15 consisting of a set of baffles 19. The roles of these compartments 8 and 9 and mixing means 15 will be discussed further later. The second embodiment is shown in FIGS. 3 and 4. This is an embodiment substantially identical to the preceding embodiment, but in which two fluidic channels which are in parallel with each other are represented. This device is referenced 100 even if all the other elements have the same reference numbers as those previously used. FIG. 3 is substantially identical to FIG. 1 in that the piston 18 is in the rest position, that is to say that it is in the lower position within the cylinder 17 and that the actuator or the manipulator moves said piston 18 through the connecting means 21. In this embodiment there is therefore an inlet 4, a single channel 3 on the downstream part, which then splits into two channels of identical section which will go up parallel to the along the body 2 of the device 100. Along each of the channels 3, so we will find as was the case previously a first compartment 8, a mixing means 15 and a second compartment 9. But in addition, there is upstream a third compartment 10. For practical reasons, a siphon is formed between the second and third compartments 9 and 10, namely that the first bend of the siphon is located above the first compartment 9 and the third compartment 10 is located at the second level elbow of the siphon. Alternatively, the compartment 10 can be deleted and the reading performed directly in the compartment 9. The role of the compartment 9 or 10 is first of all to secure any unwanted movement of the liquid towards the lower part of the card, said card being used in an upright position, and then, to increase the volume of liquid that can be used for the fluorescence detection by maximizing the diameter of the reading cap, made in the associated heating block in the instrument and position in relation to the compartment 9 or 10. Another characteristic of this embodiment is that for an inlet 4, there are two outlets 5 since each channel 3 is connected upstream to a discharge suction means 7. Thus each channel 3 is associated with a cylinder 17 and a piston 18. In this particular embodiment the two pistons 18 are independent of each other, as can be seen in FIG. 4, that is to say that one can actions according to Fl and F2 independently of one another.

The individualized two-piston configuration, with movement independent of each other, makes it possible to correct the positioning defect of the liquid segments in each fluid channel individually, in particular by a registration of said segments in the detection zone, for example, the offset that may be due to a non-homogeneous viscous sample, a slight molding defect in the fluid circuit of the board or an undesired displacement due to a thermal gradient momentarily causing a differential pressure on either side of the fluidic segment. This therefore makes it possible to increase the robustness of operation of the device. This system works as soon as there is more than one channel, using position sensors placed at appropriate places, on the one hand within the device, and on the other hand with respect to the card. according to the invention. Note that it is particularly advantageous to have an anti-extraction system of each piston 18. And advantageously, each piston 18 may be provided with a guide 23 which is connected at one end to the upper end of the connecting rod. piston 18 and at its other end, not shown in the figures, a form of larger section prohibiting the accidental extraction of the post-amplification piston during handling by the operator (eg unloading consumable instrument) at the end of the analysis, or error of positioning of the pistons by the instrument). This anti-extraction system is of course adaptable to all the embodiments envisaged by the present invention.

FIGS. 5 and 6 show a third embodiment which is substantially identical to the previous embodiment, except that it comprises in parallel eight fluidic channels 3. As for the two previous embodiments, there is only one input 4 located at a tip 16 from which a single channel 3. It divides in 2 to 3 times which gives a total number of eight channels 3 at the active part of the device 200 and therefore eight outputs 5 Each channel 3 then consists of: • a first compartment 8 followed by a mixing means 15 composed of a number of baffles 19, • a second compartment 9, and finally • a third compartment 10. Each channel ends well understood to a discharge suction means 7 constituted, as usual, by a cylinder 17 and a piston 18. In the same way as the second embodiment, this third embodiment comprises pistons 18 which are manipulable by Inter mediate the connecting means 21 independently and independently from other pistons 18 adjacent. FIG. 6 shows a longitudinal section along the axis AA of FIG. 5, which makes it possible to better visualize the connection that exists between the channel 3 and the discharge suction means 7. This section makes it possible to see the body 2 the device 200, which has on its upper surface a channel 3 delimited with respect to the outside by means of a partition film 14 made of a suitable material. The film is preferably BOPP (Bi-oriented PolyPropylene) with a silicone adhesive, but can also be made of PP (PolyPropylene), PET (Polyethylene Terephthalate), TPE (ThermoPlastic Elastomer), PP / PE-type complex films that can be sealed by laser process around the fluidic channels. This channel 3 leads to a transverse hole 25 which opens into the cylinder 17. Within this cylinder 17 is present the piston 18, the piston head 26 forms a seal with the liner of said cylinder 17. The head of the piston 26 may be composed of two parts (body with groove and elastomer O-ring) or consist of a one-piece piston, in one piece, simplifying assembly operations of the card and to reduce the cost per test. It is therefore obvious that when the actuator or the manipulator exerts a force, according to F2 on the piston 18, it will allow the suction of the gaseous or liquid fluid present in the channel 3, while the action according to F1 will allow to bring out this fluid through the outlet 4, not shown in Figure 6. There are two different ways of making integrated pistons. It can be a simple piston with or without segment ("o-ring" in English), as is the case above, or a piston with double section. Although this type of piston is well known to those skilled in the art, the advantages of using a double section piston are as follows: The intrinsic positioning accuracy is improved over a single piston (having the same diameter ) because the volume of pumped liquid is determined / calibrated by the difference in volume between the two sections of said piston (the suction is located between the two diameters of the piston).

After the pumping action, the piston is completely pushed into the shirt / cartridge where it is moved. Therefore, there is no risk that the piston is extracted by Inadvertance by the user. FIGS. 7 to 14 show the second embodiment of FIGS. 3 and 4 during the use of the device 100. It should be noted that this device 100 is very slightly different from that already exhibited since the two pistons are, in the case present, secured to each other so that the fluidic motion in each of the two needs. two pistons is deformable without physically. In Figure 7 it is noted that the liquid and biological sample 6 is included in a container 20 only although it 6 is in contact with the device 100; in particular the tip 16 is partially immersed in said liquid 6. For its part, the device 100 comprises in addition to what has been shown in Figures 3 and 4 thermostable constituents 12 present in the first compartment 8 and non-thermostable constituents In fact, the constituents are stored in solid form, for example according to the technical information disclosed in EP-B-0,641,389, to which the reader is asked to refer for more details on this subject.

It is obvious that one can imagine that there are only two compartments 8 and 9, as is the case in Figures 1 and 2. In this case, it is necessary to have constituents not thermostable and detection 13 and 14 which are present together in the second compartment 9. Similarly, if an amplification having only thermostable constituents is used, it is also possible to have all the ingredients present in a single compartment, 8 or 9. Preferably, the nozzle is provided with a pipetting cone 27, as shown in FIG. 1, for example 50 to 200 μL of capacity or else, and this is not shown in the figures but is well known to those skilled in the art, a polyethylene straw which can be thermally sealed, after completion of all the fluidic sequences in the card. In Figure 7, the container of the sample channels 3 is identical. This is of course an option and we can predict that the pistons can be detached from each other, either automatically by the controller, or manually by the manipulator in function It is also possible that the bridge connecting them to be cut biological can mount in contact with the tip, or it will be the map that will be moved by the instrument to come into contact with the liquid in the container, the latter case corresponding to the case of architectures for high speed machines. The device will then be used as a conventional pipetting cone and will therefore move along the X, Y and / or Z axes by a robot to collect in the container or containers.

In Figure 8, the connecting means 21 is moved along F2 so that the pistons 18 suck the liquid sample present in the container 20 within the card. At the end of this step, not shown in this figure, the device 100 has been raised while the pistons 18 continue their aspiration, which explains the presence of two columns of liquid in each of the channels 3. Note that the volume sucked is related to the section of the piston integrated in the card and to the length of displacement of the piston. In addition to the possible initial stop at the pipette cone, the first stop of each liquid sample 6 is at the first compartment 8, that is to say at the thermostable components 12, which allows to dilute said components that are actually stored in lyophilized or dried form, as is the case for the other components 9 and 10. The liquid segment is maintained on the position of the reagent to facilitate resuspension of the dried or lyophilized reagent, typically ten seconds, usually less than a minute. According to Figure 9 now, the movement of the fluid continues along F4 towards the mixing means 15. This movement according to F4 corresponds to the suction F2 of the piston. When the mixture 6 + 12 has reached the baffles 19, a reciprocating movement according to F3 and F4 is generated by a movement of the pistons along F1 and F2, to allow a more or less rapid passage and this on multiple occasions of said mixture within the mixing means 15. Typically the number of round-trip mixing in the map is between one and ten round trips, typically five round trips to ensure satisfactory homogenization of the reaction compounds. The changes of direction due to the presence of the baffles 19 thus allow a good mixture of the diluted constituents, said thermostable constituents 12, within the liquid sample of interest 6. By thermostable constituents 12, amplification primers are especially understood the detection probe (s), the nucleotides and any other thermostable ingredients necessary for the elongation of the primers during the amplification. In Figure 9 it is noted that there is a first zone for heating 11 represented by dotted lines. This zone symbolically represents the place where the manipulator or the automaton will come to apply a source of heat in order to proceed with the treatment of the liquid sample 6 + 12, that is to say of the sample 6 in the presence thermostable constituents 12, in order to start an amplification technique, such as NASBA. When this is done, Fig. 10 shows that the suction along F2 continues, so that the liquid columns move up to the second compartment 9 where the non-heat-stable components 13 are in turn diluted. This movement according to F4 is always due to the rise of the pistons according to F2. Non-thermostable constituents essentially mean the enzymes necessary for amplification. These include, in the context of a posttranscriptional amplification of AMV-RT (Avian Myeloblastosis Virus-Reverse Transcriptase), RNase H and T7 polymerase (DNA dependent RNA polymerase). For all liquid displacement steps, the flow rate is typically 1 μL per second. Advantageously, an optical sensor of the instrument, not shown in the figures, positioned approximately two millimeters before each reagent, embedded in the card, makes it possible to detect the presence of the liquid segments and thus to guarantee a sufficient robustness of operation by compensating for the effects related to the difference of each sample. Alternatively, the use of optical sensors also makes it possible to measure the length of the liquid segment and therefore to verify the accuracy of the liquid division performed during the suction step and loading of the sample in the card. In FIG. 11 now, each column of liquid is then descended along F1 at the mixing means 15. At this level, the whole of the mixture 6 + 12 + 13 is also driven according to F3 and F4, so that the baffles 19 allow a suitable mixture of the thermostable constituents 12 and non-heat-stable 13 within the sample 6. The arrival in the chambers can be advantageously detected by the fluorescence read head integrated into the instrument to perform the real-time measurement of the amplification reaction. FIG. 11 shows, like FIG. 9, a second zone for heating 22 which also makes it possible to carry out NASBA amplification. Finally, in FIG. 12, the suction according to F2 allows the liquid columns 6 + 12 + 13 to go up to the third compartment 10, which then acts as a read zone. It should be noted that this reading zone can be either the first 8, the second 9 or the third compartment 10. In this case, the third compartment 10 acts as a buffer zone to prevent any inappropriate rise in liquid to the at the top of the device, this action is reinforced by the closing of the valve 27. If against the third compartment 10 acts as reading area, the suction F2 is more important which allows the columns of liquid 6 + 12 + 13 to go up to said third compartment 10, where the reading can take place. Still according to another embodiment, it is possible to provide that the thermostable constituents 12 are split into two spheres, called "pellets" in English. A first sphere, present in the first compartment 8, containing the amplification primers, nucleotides and any other thermostable ingredient necessary to allow the elongation of the primers during amplification. A second sphere, present in the third compartment 10, containing the detection probe or probes necessary for the detection of the amplicons, after the amplification step. In this mixture, will have to return to the level of the mixing means 15, in which the entire mixture is still driven according to F3 and F4, so that the baffles 19 allow an appropriate mixture of thermostable constituents 12 and non-thermostable 13 within 6. The reading can be done in any of compartments 8, 9 or 10.

A final step exists, not shown in the figures, which is, immediately after the end of the positioning in the third compartment, to close the valve 27 with an actuator built into the instrument. Alternatively, said valve can be removed and replaced by a straw, as mentioned above, which can be sealed, for example to heat, valve which will then have two functions, first pipetting into the sample container and then closing by using a heating wire within the associated instrument.

EXAMPLE: This example shows the quantization performance obtained with a disposable device according to the second embodiment of the invention, namely the card with two fluidic channels in parallel (see FIGS. 3 and 4). The tests were conducted using the Human Immunodeficiency Virus (HIV) as a target. Our invention has been compared to an already commercialized product, called Nuclisens EasyQ Analyzer (Ref 285060, bioMérieux S.A., Marcy, Etoile, France), using the same biological samples containing synthetic HIV targets. 1 - Preparation of the targets: The transcripts were introduced into the two Nuclisens EasyQ devices and according to the invention. There is no sample preparation step, such as extraction. 2 - Materials and methods: The EasyQ experiments were carried out using the bioMérieux HIV2.0 kit (hereinafter referred to as PVB1) (Ref 285033, bioMérieux BV, Boxtel, The Netherlands), following the instructions for use. . This contains: • a mixture of enzymes: a sphere of enzymes, hereinafter called ENZ (batch number: 83281SXX + 45) iL enzyme diluent (83301AXX), and • a mixture of primers and of probes, hereinafter referred to as P / B: there are actually two P / B spheres (Batch No.: 83283SXX), to which are added 180 μL of 2X P / B diluent (83272AXX). NASBA with 5 μL of ENZ mixture and 20 μL of P / B + 15 μL of targets.

The disposable device according to the invention is completely automated to allow amplification and detection. It makes it possible to take the sample of the biological sample to be tested, containing the targets.

The experimental protocol of the device according to the invention is defined so that the reagents (primers, probes and enzymes in particular) have the same concentration as in the EasyQ protocol. Nevertheless the volume per test used in our invention is 5 pL instead of 40 pL with EasyQ. The quantity of reagents is thus divided by eight.

The reagents from the PVB1 kit were lyophilized and put into the device according to the invention. The lyophilization bench is associated with a Hamilton pipettor robot (Order 202997, Bonaduz, Switzerland) which allows the reproducible deposition of droplets of 1 μL for P / B and 1.25 μL for ENZ in the dedicated compartments of the inventive device.

The amplification and detection instrument used with the invention performs all functions necessary to obtain an amplification curve, i.e., sample aspiration, reagent mixing, heating and fluorescence readout. This concept removes most of the mandatory manual steps with EasyQ.

Table 1 below shows the main differences between EasyQ and the invention. EasyQ Invention Volume 40 μL 5 μL P / B 20 μL 1 μL (incorporated) ENZ 5 μL 1,25 μL (incorporated) Volume 15 μL 5 μL of sample used Steps • dilute the tubes, load the tube • and • add primers in the analyze and probes to the eluate, start. transfer to the incubator, • add the enzyme solution to the cap, • close the tube with the cap • centrifuge, • vortex, • centrifuge again, • load the tube for analysis and start. Number of up to 48 tests 2 with the tests / series device proposed Table 1: Comparison of the technical data and the method implemented by the device of the state of the art (EasyQ) and the invention As said above, the biological sample used for this study contains HIV targets. This sample is then diluted for EasyQ experiments and the invention, but the same dilution series are used. Table 2 below shows the number of HIV targets per test as well as the number of experiments performed with each of the two devices (EasyQ and invention). The pre-extraction "equivalent" copy number is the number of copies that would be required prior to an extraction step to obtain the number of copies per test (i.e. extraction equals number of copies per test divided by the extraction yield). Number of copies of 0 3.75 7.5 15 22.5 30 300 3000 the target per test (EasyQ and invention) Number of copies of 0 12.5 25 50 75 100 1000 10000 "Equivalent" pre-extraction by test Number of replicates 15 12 12 _ 12 15 15 with EasyQ 15 Number of replicates 8 8 8 4 4 4 4 4 the invention Table 2: Number of HIV targets per test as well as the number of experiments performed with each of the two devices (EasyQ and invention) By "replicate" means the number of times that the test has been run in parallel from the same starting sample. The number of copies for internal control purposes is 290 per test, both for EasyQ and for the invention.

It should be noted that the invention, in this case, allows only two tests in parallel, therefore many replicates is considerably lower with our invention than with EasyQ. The detection limit claimed for EasyQ is 25 copies (preextraction equivalent), which corresponds to 7.5 copies per test.

The data acquired with EasyQ were processed with EasyQ Director software (BioMérieux S.A., La Balme, France), using the HIV-lDB 2.0 test protocol (Ref 285033, bioMérieux B.V., Boxtel, The Netherlands). Each amplification curve, measured with the instrument using the invention, was processed using an internal tool called EDrecalc recalculation on the algorithm for calculating and interpreting the amplification curves, which is included in the EasyQ Director commercial software cited above, using the same algorithm used by the EasyQ Director software and the HIV-lDB 2.0 test protocol. 3 - Results: The raw fluorescence curves are shown in FIG. 13 for the device according to the state of the art (EasyQ) and in FIG. 14 for the invention. The amplification curves obtained with the invention are very similar to those obtained with EasyQ. A broad distribution of the fluorescence values for the fluorescence plateau of the signal from the calibrator can be observed. Since this distribution exists with each instrument, this propagation is not related to the instrumentation. 3.1 Detection limit: Due to the small number of replicates, it is not possible to determine the limit of detection with a narrow confidence interval. Thus, in Table 3 below, we have only compared the number of positive results obtained with the two instruments for some copy number values extracted from Table 2: Input values (pre-0 12.5 25 50 75 " equivalent »copies per test) EasyQ 0/15 6/12 12/12 14/15 12/12 Number of positives / number of experiments Invention 0/8 5/8 8/8 4/4 4/4 Number of positives / number of experiments Table 3: Detection limit with each of the two devices (EasyQ and invention) The limit of detection claimed for the NucliSENS HIV 2.0 test is 25 copies (detection limit at 95% positive).

With this input value, all the tests carried out with the device according to the invention are positive. With 12.5 copies, about 50% of the tests are positive with EasyQ and slightly more with the invention. We can therefore legitimately consider that our invention has results at least similar to the detection limit of the state of the art, EasyQ. 3.2 - Quantization performance: Figure 15 shows the Qratio (quantization variable) as a function of the number of copies in input. The distribution of points corresponding to the data is similar for both instruments. Using the parameters related to the lot of reagents, a person skilled in the art can obtain by calculation the number of copies, the average of which is plotted in logarithmic scale in FIG. 16. The following tables 4 and 5 show the performance of the qualification associated with these two instruments Log data (input) average precision input accuracy 25 1, 40 1, 31 0, 19 0, 09 50 1.70 1.40 0.20 0.30 75 1.88 1.64 0.19 0.24 100 2.00 1.70 0.23 0.30 1000 3.00 2.75 0.14 0.25 10000 4.00 3.83 0.07 0.17 Table 4: Performance of qualification for EasyQ Log data (input) average input precision precision 25 1.40 1.47 0.18 0.07 50 1, 70 1, 34 0, 33 0, 36 75 1, 88 1, 77 0, 05 0, 11 100 2.00 1.82 0.34 0.18 1000 3.00 2.89 0.21 0.11 10000 4.00 3.84 0.13 0.16 Table 5: Performance of qualification for the invention

According to the high-level specifications of the HIV2.0 test, the precision, ie the standard deviation of the results of different replicates, must be less than 0.3 log. Some precision values for the instrument prototype data associated with the device are above this specification. This may be due to the small number of replicates that does not give a correct estimate of accuracy. The prototype complies well with the accuracy specification (that is, the difference between the result and the number of copies at the input of the test) is 0.25.

The linearity for the invention is the same as for EasyQ but needs to be measured beyond the 104 copies of this study. 4 - Conclusion: The present invention, in its configuration with two parallel fluidic channels was used to detect HIV targets via the HIV v.2.0 kit. The results were compared with the EasyQ analyzer, which was used as a reference.

The performances of the invention are in line with the most important constraints necessary for carrying out an HIV test (HIV v.2.0) and are at least comparable to those recorded with the EasyQ analyzer.

Because the invention used a reaction volume of 5 μL (instead of 40 μL for EasyQ), the amount of reagents per test was divided by eight. This leads to a much lower cost of production per test, because this cost is mainly due to the enzymes, representing about 80% of all the ingredients of the kit. The device according to the invention also greatly reduces the number of manual steps, since only three basic actions are necessary to launch a test: • loading of the device according to the invention, • to load the tube containing the extracted targets (by the device EasyMAG Extraction System (Ref 200111, bioMérieux SA, Marcy l'Etoile, France), and • start the test.5 REFERENCES

1 - Disposable device 2 - Solid body of the device 1 3 - Circuit or fluidic channel within the body 2 4 Hole opening from the called channel inlet 5 - Hole opening out of the channel called exit 6 - Liquid and biological sample of interest 7 Medium of suction-discharge 8 First compartment along the canal 3 9 Second compartment along the canal 3 10 Third compartment along the canal 3 11 First zone for heating 12 - Thermal constituents contained in compartment 8 13 - Non-thermostable constituents contained in the compartment 9 14 Film delimiting 15 Mixing means 16 Integrated pipette cone or cone receiving mandrel 28 17 - Cylinder of the suction-discharge means 7 18 Piston of the suction-discharge means 7 19 Baffle of the mixing means 20 20 - Container where the sample 6 is initially present 21 Connecting means with an actuator 22 Second zone for heating 23 Piston guide 18 25 - Transve hole rsal 26 Piston head 27 - Final closing or sealing valve 28 Conventional pipette cone 100 - Disposable device with two parallel 3 channels 200 - Disposable device with eight parallel channels 3 F1 - Movement of the piston 18 in the cylinder 19 which decreases the volume of fluid within the fluidic circuit 3 F2 Movement of the piston 18 in the cylinder 19 which increases the volume of fluid within the fluidic circuit 3 F3 Movement of the sample 6 under the action of the movement F4 - Movement of the sample 6 under the action of the movement F5 Fitting of the cone 28 on the sleeve 16 F1 F2

Claims (20)

REVENDICATIONS1. Disposable device (1) for amplifying at least one target nucleic acid, present in a liquid and biological sample of interest (6), which consists of a solid body (2), at least one channel fluidic means (3) connecting an inlet (4), by which all or part of the sample of interest (6) can be sucked and / or discharged, and an outlet (5), which is itself connected to a means suction-discharge device (7) of said sample of interest, the fluidic channel (3) further comprising the inlet (4) to the outlet (5). A first compartment (8) containing all or part of the thermostable constituents (12) necessary for carrying out the amplification; means for mixing (15) the constituents (12 possibly associated with 13) with the sample of interest (6), • a second compartment (9) containing all or part of the non-thermostable constituents (13) necessary for carrying out the amplification, • and in addition at least one zone intended for heating (11) said sample of interest (6) mixed with said amplification components (12 associated with 13) to allow amplification of the target nucleic acid. 2. Device according to claim 1, for the detection of amplicons, characterized in that it further comprises at the second compartment (9), all or part of the detection components necessary for the detection of amplicons. 25 3. Device according to claim 1, for the detection of amplicons, characterized in that it further comprises at the level of the fluidic channel (3) a third compartment (10) containing all or part of the constituents necessary for the detection of the amplicons. amplicons. 4. Device according to claim 3, characterized in that the third compartment (10) is located between the second compartment (9) and the outlet (5) of the device (1). 5. Device according to any one of claims 1 to 4, characterized in that the inlet (3) receives a cone (28) pipette (16) or the tip (3) pipette 15 has a configuration in the form of a pipette cone. 6. Device according to any one of claims 1 to 5, characterized in that the suction-discharge device (7) is of the piston type such as for example a pipette (7). 7. Device according to any one of claims 1 to 6, characterized in that the section of the channel (3) is constant and the compartments (8, 9 and 25 possibly 10) are of a larger section. 8. Device according to any one of claims 1 to 7, characterized in that the inlet (4) is in relation to at least two fluidic channels (3). 9. Device according to any one of claims 1 to 8, characterized in that the outlet (5) is constituted by at least two fluidic channels (3). 30 10. Device according to any one of claims 1 to 9, characterized in that the constituents (12 & 13) are formed of freeze-dried or dried biological compounds, soluble in the sample of interest (6). 11. Device according to any one of claims 1 to 10, characterized in that the suction-discharge means (7) is an integral part of the disposable device (1). 10 12. Device according to claim 11, characterized in that the suction-discharge means (7) consists of a cylinder (17) connected to the fluidic channel (3) and a piston (18) movable within of the cylinder (17) 15 manually or via an actuator. 13. Device according to any one of claims 1 to 12, characterized in that the mixing means (15) consists of the fluid channel (3), the path 20 comprises at least one baffle (19). 14. A method of amplifying at least one target nucleic acid, present in a liquid and biological sample of interest (6), carried out within a device according to any one of claims 1 to 13, which comprises: (a) drawing all or part of the sample of interest (6) through the inlet (4) into the device (1), (b) moving said sample (6) to dissolve the constituents of the sample (6). thermostable amplification (12), (c) mixing sample (6) and thermostable constituents (12), (d) applying a first temperature gradient to denature the nucleic acid of interest, (e) displacing the mixture ( 6 + 12) for dissolving non-thermostable amplification constituents (13), (f) mixing mixture (6 + 12) and non-thermostable constituents (13), and (g) applying at least one second temperature gradient in order to amplify the denatured nucleic acid. 10 15. Method according to claim 14, characterized in that the thermostable amplification components (12) of step (b) also contain restriction enzymes, which are not necessarily heat-stable, but which allow the digestion of the acids. Nucleic acids of interest, which are deoxyribonucleic acids (DNA), prior to application of the first temperature gradient of step (d). 16. Method according to any one of claims 14 or 15, allowing the detection of amplicons, characterized in that it consists, after step (g) to (h) mix mixture (6 + 12 + 13) (i) moving the new mixture (6 + 12 + 13) to dissolve the detection components (14), and (j) detecting the presence of amplicons. 17. Method according to any one of claims 14 to 16, characterized in that the amplification is a PCR amplification, for which the first temperature gradient is between 90 and 100 ° C, and the second temperature gradients is an alternation of the temperature in three different stages: between 90 and 100 ° C for the first denaturation temperature, preferably around 94 ° C, between 50 and 60 ° C for the second hybridization temperature, preferably about 55 ° C, between 70 and 75 ° C for the third polymerization temperature, preferably about 72 ° C. 18. Method according to any one of claims 14 to 16, characterized in that the amplification is a post-transcriptional amplification (NASBA or TMA), for which the first temperature gradient is between 60 and 70 ° C, preferentially about 65 ° C, and the second temperature gradient is between 40 and 50 ° C for the second polymerization temperature gradient. 19. Method according to any one of claims 14 to 18, characterized in that the first temperature gradient is applied at the level of the first compartment (8) and / or the mixing means (15), and that the second temperature gradients is or are applied at the mixing means (15) and / or the second compartment (9) and / or the third compartment (10) 20. Process according to any one of claims 14 to 18, characterized in that the first temperature gradient is applied for 5 to 20 minutes, preferably 15 minutes, and that the second temperature gradient or gradients is or are applied. in the case of a PCR amplification: for the denaturation, for less than one minute, preferably from 2 to 20 seconds, preferably 5 seconds, for hybridization, for less than one minute, preferably from 2 to 20 seconds, preferentially seconds, and - for the polymerization, for less than two minutes, preferably from 5 to 80 seconds, preferably 10 seconds, • in the case of a posttranscriptional amplification, for less than two hours, preferably from 5 to 80 minutes, and again more preferably: about 60 minutes in the case of RNA target nucleic acids, or about 90 minutes in the case of target nucleic acids DNA. 15